1. Field of the Invention
This invention relates to one-vial methods and kits for radiolabeling a protein with a radiometal ion of a radionuclide that binds tightly to sulfhydryl groups by generating sulfhydryl groups on the protein using a tertiary thiol-containing chelating agent. Use of a tertiary thiol-containing chelating agent provides a protein containing a t-thiol-containing derivative that can be deprotected to generate free sulfhydryl groups which then are labeled with a radionuclide.
2. Description of Related Art
It is known that certain radiometals bind tightly to sulfur ligands, including, e.g., Tc-99m from reduced pertechnetate, Re-186 and Re-188 ions, Cu-67 ions, Hg-197 ions and Bi-212 ions. Some of these radiometals have been bound to proteins, especially antibodies or antibody fragments. Technetium-99m is an ideal radionuclide for scintigraphic imaging because of its nuclear properties. Technetium-99m has a single photon energy of 140 KeV, a half-life of about 6 hours and it is readily available from a .sup.99 Mo-.sup.99m TC-generator.
The element below technetium in the periodic table, rhenium, has similar chemical properties and can be labeled to protein using similar techniques. There are some 34 isotopes of rhenium and two of them in particular, rhenium-186 (t.sub.1/2 90 hours, gamma 137 KeV, beta 1.07, 0.93 MeV) and rhenium-188 (t.sub.1/2 17 hours, gamma 155 KeV, beta 2.12 MeV) are prime candidates for radioimmunotherapy using monoclonal antibody approaches.
Two methods commonly are used to label proteins such as antibodies or antibody fragments with radiometals. The first approach is the direct labeling method whereby the radiometal is bound directly to the protein molecule. Direct labeling involves reducing the protein to generate free sulfhydryl groups and then directly attaching a reduced radionuclide to the free sulfhydryl groups. Direct labeling of protein has been accomplished using a "pre-tinning" protocol, requiring severe conditions and long "pre-tinning" times, but radiolabeling at 100% incorporation was not achieved; Crockford et al., U.S. Pat. No. 4,323,546 (see also U.S. Pat. No. 4,424,200). In this process, the presence of extremely high amounts of stannous ion for long periods compromised the immunoreactivity of the antibody. The process also generally necessitated a post-labeling purification column. Other direct labeling methods have required separate vials, one for antibody and one for stannous ion complexed to a transchelator such as a phosphate and/or phosphonate.
Another problem associated with the direct labeling method is that antibodies directly labeled with .sup.99m Tc and/or Rhenium have been reported to be unstable in vivo, i.e., a significant proportion of the radionuclide dissociates from the labeled antibody fairly quickly upon injection of the labeled antibody into the bloodstream. When labeled antibody is used for external imaging, this instability leads to accumulation of radioactivity in locations other than those at which the radiolabeled antibody localize. This reduces the resolution of the method by attenuating the localized radioactivity and by increasing the background activity due to non-specific distribution of the radioisotope. Rhodes et al., TUMOR IMAGING, Chpt 12, pps 111-24 (Mason Publ., USA, 1982), disclose that unstable antibodies directly labeled with .sup.99m Tc could be purified using an elaborate permeation chromatographic method which also complicates the method of directly radiolabeling proteins. Finally, the presence of residual technetium is difficult to remove as are the colloids it may form, and both tend to contribute to undesirable non-specific background radiation.
The second method of radiolabeling proteins is the indirect method whereby a complexing agent is coupled to the protein and the radiometal is attached to the protein via the complexing agent. The complexing agent typically contains free or protected sulfhydryl groups that are capable of complexing with the reduced radionuclide on one end and groups capable of reacting with the protein on the other end. Indirect labeling methods using conjugated chelating groups such as diethylenetriaminepentaacetic acid (DTPA) (Khaw et al., J. Nucl. Med., 23:1011-19 (1982) or a variety of sulfur/nitrogen (S.sub.2 N.sub.2) chelators such as bis-thiosemicarbozones and the like are known. Khaw et al., Science, 209:295-97 (1980) discloses antibodies to cardiac myosin labeled with indium-111 via DTPA and use of the labeled antibodies to image for myocardial infarction. See also, Krejcarek et al., Biochem. Byophys. Res. Commun., 77:581-85 (1977); Childs, R. L. and Hnatowich, D. J., J. Nucl. Med., 26:293 (1985). In a more recent approach, Fritzberg et al. J. Nucl. Med., 27:957 (1986), Baidoo et al., Cancer Research (Suppl.), 50:799s-803s, (1990) describe the use of a particular diamidodithiol and diaminodithiol groups as chelating agents.
These chelating agents include free thiol groups that may serve to reduce disulfide bonds in the protein to be labeled. Thus, labeling in accordance with the methods described in the aforementioned documents can occur either directly at the reduced disulfide bonds in the protein or on the free sulfhydryl groups on the chelating agents. These methods also typically include the use of a transchelator such as glucoheptonate to keep the reducing agent in solution. The methods disclosed in Fritzberg et al. and Baidoo et al. therefore are not practical and easy to use, and may not specifically label the chelating agent on the pendant free sulfhydryl groups of the chelating agent.
Many of the aforementioned conventional chelate-based indirect labeling techniques often involve elaborate syntheses of chelating agents, and, frequently, the need for a `pre-labeling` procedure to accomplish antibody radiolabeling. These processing steps compromise the certain qualities such as ease of use, and practicality typically desired in the diagnostic industry. Other indirect labeling methods involve the thiolation of proteins; Wong, S. S., in CHEMISTRY OF PROTEIN CONJUGATION AND CROSS-LINKING, CRC Press, Inc., Boca Raton, Fla., pp 17-23 (1991). Methods using ring-opening of iminothiolane such as those described in McCall, M. J., Diril H., and Meares, C. F., Bioconjugate Cem. 1:222-26, (1990) and Goff, D. A. and Carroll, S. F., Bioconjugate Chem. 1:381-86 (1990), have the disadvantage of continually generating thiols, resulting in the formation of aggregates, thus necessitating simultaneous derivatization of generated thiols. The use of N-acetylhomocysteine thiolactone also is expected to result in similar problems. S-acetylmercaptosuccinic anhydride is often employed as a thiolating agent, in a reaction with lysine residues of proteins, with the thiol acetate converted to thiol in a subsequent step. Use of this reagent, however, results in an alteration of the overall charge ratio of the thiolated protein, which has implications for the conformation and pharmacokinetics of the antibody, Wong et al., supra. Other reagents, such as (3-acetylthio propionyl)-thiazolidine-2-thione, will generate, after deprotection, a mercapto group which is attached to an unsubstituted methylene group.
Dean, U.S. Pat. No. 5,180,816 discloses a method of radiolabeling an antibody with technetium whereby a chelating agent containing a primary thiol group is used to attach to free sulfhydryl groups on the antibody. Hence, the antibody first is reduced and cleaved to fragments to generate free sulfhydryl groups, or alternatively, the protein is thiolated with, for example, 2-iminothiolane to generate free sulfhydryl groups. A chelating agent containing a protected thiol group at one end then is used to attach to the free sulfhydryl groups on the antibody at the other end, and is subsequently deprotected and contacted with technetium to effect radiolabeling. The process of Dean involves a disadvantageous cleavage of the antibody which may result in excessive fragmentation or destruction of the antigen binding site on the antibody.
Indirect labeling of a protein without generation of free sulfhydryl groups, i.e., by attachment of the chelating agent to, for example, amino functionalities on the protein, is not possible with Dean's primary thiol-containing chelating agents even though Dean mentions that a N-hydroxysuccinimide ester may be used to bind the chelating agent to the amine functions on the protein. The reason for this is that the protein amino groups react not only with the N-hydroxysuccinimide carboxylic esters, but they also cleave the protected primary thiol group at the opposite end of the chelating agent. This cleavage results in premature deprotection and oxidation of the generated free thiol groups and prevents efficient and quantitative labeling of the antibody.
Other problems associated with the use of chelating agents is that one-pot labeling where the protein-chelating agent conjugate is contacted with tin prior to reaction with the radionuclide is not possible. The reason for this is because the presence of the tin reduces the disulfide bonds in the protein thereby destroying the chelator-specificity of the radiolabel and possibly destroying the efficacy of the protein. In addition, many chelating agents when used in immunotherapy using rhenium as the radionuclide often accumulate in toxic amounts in the kidney. For imaging purposes, many radiolabeled proteins, for example, whole IgG, radiolabeled with technetium, tends to clear to and congregate in the liver thereby adversely affecting the efficiency of the imaging since many tumors can be metastasized throughout the body. Also, the tumor to non-tumor ratio for many conventional radiolabeled proteins tends to be too low thereby advserely affecting the efficiency of the image.
Thus, there exists a need to develop a method of labeling a protein using a chelating agent that is capable of forming a complex with a non-antigenic binding site on a protein that has not been reduced or thiolated, and that is capable of forming a complex with a radionuclide so that the radiolabeled protein is not further cleaved when contacted with tin and subsequently frozen or lyophilized. There also exists a need to develop a method of labeling a protein by using a bifunctional chelating agent containing pendant protected thiol groups whereby the reactive groups on the protein will not prematurely cleave the protected thiol groups on the bifunctional chelating agent. There also exists a need to develop a method and kit that is easy to use and does not involve complicated synthesis procedures or multiple containers for the protein and reducing agent and does not involve the use of a transchelator. There also exists a need to develop a method of radiolabeling a protein for use in radioimmunoimaging or radioimmunotherapy whereby the radiolabeled protein has good tumor uptake, low kidney uptake, does not clear entirely in the liver, spreads out broadly throughout the body and provides a good tumor to non-tumor ratio for imaging purposes. There also exists a need to develop a method of radiolabeling a F(ab').sub.2 monoclonal antibody that does not subsequently cleave to Fab' even if tin is present in the one-pot labeling kit.